Over a hundred years ago Alois Alzheimer identified the clinical and pathologic hallmarks of a dementing illness that came to be known as Alzheimer's Disease (AD). (Alzheimer, et al., An English translation of Alzheimer's 1907 paper, “Uber eine eigenartige Erkankung der Hirnrinde”, Clin Anat 8 (6), 429-431 (1995)). AD is characterized clinically by progressive loss of cognition and pathologically by the accumulation of amyloid plaques, neurofibrillary tangles, synaptic loss and neuronal death. It is estimated that patients already have lost as much as 50% of their neurons at the time of their first clinical symptoms, supporting the relevance of neuronal death in the disease. (DeKosky, et al., Revision of the criteria for Alzheimer's disease: A symposium, Alzheimers Dement 7 (1), e1-12 (2011)). In the century since Alzheimer's studies, β-amyloid and tau were identified as the protein components of plaques and tangles respectively, and genetic studies of familial AD identified mutations in genes regulating the production of Aβ, supporting a critical role for Afβ in the disease. (Li, et al., beta-Amyloid protein-dependent nitric oxide production from microglial cells and neurotoxicity, Brain Res 720 (1-2), 93-100 (1996); Kruman, et al., Evidence that 4-hydroxynonenal mediates oxidative stress-induced neuronal apoptosis J Neurosci 17 (13), 5089-5100 (1997); Pike, et al., Beta-amyloid neurotoxicity in vitro: evidence of oxidative stress but not protection by antioxidants, J Neuroehem 69 (4), 1601-1611 (1997); Keller, et al., Mitochondrial manganese superoxide dismutase prevents neural apoptosis and reduces ischemic brain injury: suppression of peroxynitrite production, lipid peroxidation, and mitochondrial dysfunction J Neurosci 18 (2), 687-697 (1998); and Guo, et al., Increased vulnerability of hippocampal neurons from presenilin-1 mutant knock-in mice to amyloid beta-peptide toxicity: central roles of superoxide production and caspase activation, J Neurochem 72 (3), 1019-1029 (1999)). More recently, Caspase 2 has been identified as a key factor in the role played by Afβ in AD and in neuronal dysfunction generally. (Reviewed in, Troy and Ribe, Caspase 2: Vestigial Remnant or Master Regulator?, Sci. Signal., 1 (38), e42 (2008)).
The significance of Caspase 2 as a critical mediator of neuronal dysfunction and death in AD is supported by several lines of evidence. First, brain tissue from patients with mild and severe AD shows increased expression of Caspase 2 when compared to age-matched controls (FIG. 1). Similarly, the neurological deficits of J20 hAPP mice, which exhibit age-related spine loss and cognitive dysfunction, can be blocked when the mice are engineered to lack Caspase 2 (FIG. 2). (Pozueta, et al., Caspase 2 is required for Abeta induced spine loss, American Soc. for Cell Biology Annual meeting (2010)). A further connection between Caspase 2 and AD is provided by studies of CUGBP2, a gene product that has been linked to late-onset AD. (Wijsman, et al., Genome-wide association of familial late-onset Alzheimer's disease replicates BIN1 and CLU and nominates CUGBP2 in interaction with APOE PLoS Genet 7 (2), e1001308 (2011)). Specifically, it has been found that CUGBP2 is induced by Aβ42 in a Caspase 2-dependent manner and is required for Aβ42 mediated death (FIG. 3). Taken together these data provide compelling evidence for an Aβ-induced Caspase 2-mediated pathway of neuronal dysfunction in AD.
Caspase 2 contains a long prodomain with a “caspase recruitment domain” (CARD). Activation of Caspase 2 requires dimerization via the CARD. 0 (RIP-associated ICH-1/CED-3-homologous protein with a death domain) contains a CARD and has been shown to function as an adaptor for Caspase 2. In non-neuronal cells, phosphorylation of Ser-140 in the prodomain of Caspase 2 has been shown to inhibit Caspase 2 activation. (Nutt, et al., Metabolic regulation of oocyte cell death through the CaMKII-mediated phosphorylation of Caspase 2, Cell 123 (1), 89-103 (2005); and Shin, et al., Caspase 2 primes cancer cells for TRAIL-mediated apoptosis by processing procaspase-8, Embo J 24 (20), 3532-3542 (2005)). Presumably such inhibition is achieved by blocking the interaction between Caspase 2 and RAIDD.
The activation complex for Caspase 2 has been proposed to be the PIDDosome, comprised of Caspase 2, RAIDD and PIDD. (Tinel, et al., The PIDDosome, a protein complex implicated in activation of Caspase 2 in response to genotoxic stress, Science 304 (5672), 843-846 (2004)). However, two independent studies of different lines of PIDD null mice suggest that non-neuronal death does not require PIDD. (Manzi, et al., Caspase 2 activation in the absence of PIDDosome formation, J Cell Biol 185 (2), 291-303 (2009); and Kim, et al., DNA damage- and stress-induced apoptosis occurs independently of PIDD, Apoptosis: an international journal on programmed cell death 14 (9), 1039-1049 (2009)). In addition, when Pen1-siRNAs capable of effectively targeting the destruction of PIDD and RAIDD mRNAs are employed, knockdown or knockout of PIDD does not block Caspase 2-dependent death (FIGS. 4 and 5). Thus, while PIDD is not critical for activation of Caspase 2 in neurons, RAIDD is required for Caspase 2-dependent neuronal death.
There remains a need in the field for compositions capable of robust and specific inhibition of Caspase 2-associated neuronal dysfunction. The present invention addresses this need by the development of a novel inhibitor of the Caspase 2/RAIDD interaction, and membrane permeable complexes thereof, which function to inhibit Caspase 2 activation.